Approval Sheet Single-molecule Studies of DNA Mismatch Recognition Open Access

Abstract

Single-molecule Studies of DNA
Mismatch Recognition

The DNA mismatch repair system protects the genome from spontaneous
mutations by recognizing and repairing DNA synthesis errors in a pathway that is highly
conserved. The MutS family of proteins initiate DNA mismatch repair by specifically
binding mismatched or extrahelical bases and communicating the presence of damage to
downstream repair proteins in an ATP-dependent manner. Previous structural studies
have implied that MutS-induced conformational changes on DNA are central to damage
recognition. Because the conformational changes occur on the timescale of seconds, it is
difficult to obtain kinetic information on this highly dynamic process with traditional
ensemble techniques. In this work, we use single-molecule fluorescence resonance
energy transfer to investigate the conformational dynamics of mismatched DNA
substrates in the absence and presence of DNA mismatch recognition proteins. We
present quantitative kinetic information on the dynamics of DNA substrates and on the
rates of MutS binding and dissociation in a variety of buffer conditions.

Single-molecule Studies of DNA
Mismatch Recognition

B.A., Hendrix College, 2006
Advisor: Ivan Rasnik, Ph.D.
A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies
of Emory University in partial fulfillment of the requirements for the degree of Doctor of
Philosophy in Physics
2012

Table of Contents

Table of Contents
Chapter 1:

Introduction ...……………………………………………………………… 1

Figure 1.1 ………………………………………………………………………..10

Table 1.1 ……………………………………………………………………….. 11
Chapter 2: Single-molecule fluorescence resonance energy transfer ………………... 12

Figure 2.1 ………………………………………………………………………. 28

Figure 2.2 ………………………………………………………………………. 28

Figure 2.3 ………………………………………………………………………. 29

Figure 2.4 ………………………………………………………………………. 30

Figure 2.5 ………………………………………………………………………. 30

Figure 2.6 ………………………………………………………………………. 31

Figure 2.7 ………………………………………………………………………..32
Chapter 3: Discrete conformational dynamics in three-way DNA junctions ...……… 33

Figure 3.1 ………………………………………………………………………. 52

Figure 3.2 ………………………………………………………………………. 53

Figure 3.3 ………………………………………………………………………. 54

Figure 3.4 ………………………………………………………………………. 55

Figure 3.5 ………………………………………………………………………. 56

Figure 3.6 ………………………………………………………………………. 57

Figure 3.7 ………………………………………………………………………..58

Figure 3.8 ………………………………………………………………………..59

Figure 3.9 ………………………………………………………………………..60

Chapter 4:

Conformational trapping of Human Mismatch Recognition Complex
MSH2/MSH3 on repair-resistant DNA loops…………………………….. 61

Figure 4.1 ………………………………………………………………………. 83

Table 4.1 ……………………………………………………………………….. 84

Table 4.2 ……………………………………………………………………….. 84

Figure 4.2 ………………………………………………………………………. 85

Figure 4.3 ………………………………………………………………………. 86

Figure 4.4 ………………………………………………………………………. 87

Figure 4.5 ………………………………………………………………………. 88

Figure 4.6 ………………………………………………………………………. 89

Figure 4.7 ………………………………………………………………………..90

Figure 4.8 ………………………………………………………………………..91

Table 4.3 ……………………………………………………………………….. 92

Table 4.4 ……………………………………………………………………….. 92

Figure 4.9 ………………………………………………………………………..93

Figure 4.10 ……………………………………………………………………... 94

Figure 4.11 ……………………………………………………………………... 95
Chapter 5: The binding kinetics of E. coli MutS influence DNA mismatch
selectivity…………………………………………………………………. 96

Figure 5.1 ……………………………………………………………………... 112

Figure 5.2 ………………………………………………………………………113

Figure 5.3 ………………………………………………………………………114

Figure 5.4 ………………………………………………………………………115

About this Dissertation

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School	Laney Graduate School
Department	Physics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biophysics, General
Keyword	Three-way DNA junction Biophysics DNA mismatch repair Fluorescence Conformational dynamics MSH2/MSH3 MutS DNA mismatch recognition single-molecule FRET
Committee Chair / Thesis Advisor	Rasnik, Ivan, Emory University
Committee Members	Weeks, Eric, Emory University Warncke, Kurt, Emory University Berland, Keith, Emory University Doetsch, Paul, Emory University

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